Hepatitis C virus (HCV) was identified over a decade ago and is now known to be the leading cause of non-A and non-B viral hepatitis (Choo et al., Science (1989) 244:359-362; Armstrong et al., Hepatology (2000) 31:777). HCV infects approximately 3% of the world population, an estimated 200 million people (Cohen, J., Science (1999) 285:26). About 30,000 newly acquired HCV infections occur in the United States annually. Additionally, there is a large incidence of HCV infection in developing countries. Although the immune response is capable of clearing HCV infection, the majority of infections become chronic. Most acute infections remain asymptomatic and liver disease usually occurs only after years of chronic infection.
The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. At least six distinct, but related genotypes of HCV, based on phylogenetic analyses, have been identified (Simmonds et al., J. Gen. Virol. (1993) 74:2391-2399). The virus encodes a single polyprotein having about 3000 amino acid residues (Choo et al., Science (1989) 244:359-362; Choo et al., Proc. Natl. Acad. Sci. USA (1991) 88:2451-2455; Han et al., Proc. Natl. Acad. Sci. USA (1991) 88:1711-1715).
In particular, several proteins are encoded by the HCV genome. The order and nomenclature of the cleavage products of the HCV polyprotein is as follows: NH2—C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Another protein (F) has also been identified and results from translational frame-shifting within the C gene. Branch et al., Semin. Liver Dis. (2005) 25:105-117. Initial cleavage of the polyprotein is catalyzed by host proteases which liberate three structural proteins, the N-terminal nucleocapsid protein (termed core) and two envelope glycoproteins, gpE1 (also known as E) and gpE2 (also known as E2/NS1), as well as nonstructural (NS) proteins that encode the viral enzymes and other activities. The NS regions are termed NS2, NS3, NS4 and NS5. NS2 is an integral membrane protein with proteolytic activity and, in combination with NS3, cleaves the NS2-NS3 junction. The NS3 protease, along with its NS4a cofactor, serves to process the remaining polyprotein. In these reactions, NS3 liberates an NS3 cofactor (NS4a), two proteins (NS4b and NS5a), and an RNA-dependent RNA polymerase (NS5b). Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a junction, catalyzed by the NS3 serine protease.
Development of anti-viral drugs and vaccines against HCV infection has been hindered by the lack of a suitable animal model or cell culture system for HCV replication. HCV has been found to grow poorly in cell culture, and the use of subgenomic replicons, which replicate more efficiently in cultured cells, has failed to produce infectious viral particles (Bartenschlager et al. (2000) J. Gen. Virol. 81:1631-1648; Lohmann et al. (1999) Science 285:110-113; Ikeda et al. (2002) J. Virol. 76:2997-3006; Pietschmann et al. (2002) J. Virol. 76:4008-4021). Furthermore, existing replication systems have been found to be inadequate for testing some antiviral strategies. Replication systems based on self-replicating subgenomic replicons that only express nonstructural viral proteins do not emulate all steps of the HCV virus life cycle that may be targeted for antiviral intervention. Cell lines that are stably transfected with HCV precursors, but that are incapable of HCV RNA replication, cannot be used to screen for antiviral drugs that block viral RNA replication. Recently, Pietschmann et al. demonstrated that infectious virus could be produced from a bicistronic artificial HCV genomic construct in cell culture utilizing a chimeric construct comprising on one cistron, sequences from HCV type 2a strain JFH1 and sequences from a second HCV strain, and on a separate cistron, a reporter gene for monitoring transcription and infection (PNAS (2006) 103:7408-7413).
There remains a need, however, for improved tissue culture systems for production of HCV and for development of a tissue culture system that can cost-effectively and efficiently produce infectious HCV particles that can be used in testing antiviral therapeutics.